Stacey Peterson
Research Abstract
My research focuses on understanding E.coli DNA adenine methyltransferase (Dam) in its role as a bacterial gene regulator. In particular, I am studying Dam's processivity and how it is regulated by factors such as the flanking sequences surrounding the GATC recognition site, the methylation state of the DNA substrate (hemi- verses un-methylated GATC sites), and cellular conditions like AdoMet and salt concentrations. The system I am using to study Dam processivity is the pap operon. Dam is a key regulator of the pap operon, which codes for the formation of pili in uropathogenic bacteria (1). There are two GATC sites in the pap promoter separated by 102 basepairs and Dam methylates these sites fairly distributively in vitro (2). Mutating the flanking sites of each GATC to sequences previously found to be preferred by Dam (3) increases the processivity of Dam on this substrate (4). Interestingly, the GATC flanking sequences in other Dam-regulated promoters are very similar to those in the pap promoter suggesting that Dam's poor preference for such sites may be important in its role in gene regulation (4, 5), as compared to its role in replication and mismatch repair where its job is to methylate over 99% of the bacterial genome (6). I hope to gain further insights as to what modulates Dam processivity and how these modulations affect Dam's mechanistic role in gene regulation. A better understanding of Dam's mechanistic properties as a regulator of virulence genes could lead to further advancements in drug design for bacterial infections.
1. Hernday, A. D., Braaten, B. A., & Low, D. A. (2003). The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch. Mol. Cell 12, 947-957
2. Mashhoon, N., Carroll, M., Pruss, C., Eberhard, J., Ishikawa, S., Estabrook, R. A., & Reich, N. (2004). Functional characterization of Escherichia coli DNA adenine methyltransferase, a novel target for antibiotics. J. Biol. Chem. 279, 52075-52081
3. Bergerat, A., Kriebardis, A., & Guschlbauer, W. (1989). Preferential site-specific hemimethylation of GATC sites in pBR322 DNA by Dam methyltransferase from Escherichia coli. J. Biol. Chem. 264, 4064-4070
4. Peterson SN, Reich NO. (2006). GATC Flanking Sequences Regulate Dam Activity: Evidence for how Dam Specificity may Influence pap Expression. J Mol Biol. 355, 459-72
5. Tavazoie, S. & Church, G. M. (1998). Quantitative whole-genome analysis of DNA-protein interactions by in vivo methylase protection in E. coli. Nat. Biotechnol. 16, 566-571
6. Lobner-Olesen, A., Skovgaard, O., & Marinus, M. G. (2005). Dam methylation: coordinating cellular processes. Curr. Opin. Microbiol. 8, 154-160
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